cell adhesion biophysics on dynamic polymer constructs

122 Pages·2015·7 MB·English

by  Andreas Kourouklis

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Doctoral Dissertations Dissertations and Theses
March 2015
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Andreas Kourouklis
University of Massachusetts Amherst
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CELL ADHESION BIOPHYSICS ON DYNAMIC
POLYMER CONSTRUCTS
A Dissertation Presented
by
ANDREAS P. KOUROUKLIS
Submitted to the Graduate School of the
University of Massachusetts Amherst in partial fulfillment
of the requirements for the degree of
DOCTOR OF PHILOSOPHY
February 2015
Chemical Engineering
(cid:13)c Copyright by Andreas P. Kourouklis 2015
All Rights Reserved
CELL ADHESION BIOPHYSICS ON DYNAMIC
POLYMER CONSTRUCTS
A Dissertation Presented
by
ANDREAS P. KOUROUKLIS
Approved as to style and content by:
Harry Bermudez, Chair
David Hoagland, Member
Shelly Peyton, Member
John Collura, Interim Department Head
Chemical Engineering
To those who remained loyal
ACKNOWLEDGMENTS
This thesis was completed under the mentorship of Professor Bermudez. I am
grateful to him for his ongoing support and advice in academic and personal issues. I
appreciate his time, ideas and funding that made my Ph.D. experience a constructive
and stimulating journey. As characteristic of our interaction, I recollect our amusing
discussion on block copolymer thermodynamics during our flight to the spring MRS
at San Francisco.
I am also thankful to my research committee members, Professor Dave Hoagland
and Professor Shelly Peyton. They generously shared their feedback toward the
measurement of film mobility and the characterization of cell adhesion responses.
Moreover, I am grateful for their academic support toward my future professional
goals.
I would like to acknowledge the Department of Chemical Engineering, the Depart-
ment of Polymer Science and Engineering and the Graduate School of the University
of Massachusetts at Amherst for the opportunities they provided to me. I am par-
ticularly indebted to Professor McCarthy’s group for their generosity to use their
lab facilities toward the creation of supported polymer films. The measurements of
film mobility were performed at the central microscopy facility upon the constructive
comments and discussion of Dale Callaham.
In addition, I would like to thank my past and present colleagues in Bermudez
groupwithwhomIdevelopedstrongprofessionalandfriendshiprelationships. Irecall
with gratitude the days I spent with Ronald Lerum, Lang Chen, Phapanin Charoen-
phol (Ploy), Stephen Strassburg, Sandipan Dawn, Purnendu Kumar Nayak, Laura
v
Lanier, Jung Won, Adam Hathorne and Treniece Terry sharing our plans, concerns
and joys. I would like to specifically acknowledge Ronald Lerum who was an excel-
lent mentor and lab partner. Together, we worked for a couple of years toward the
development of mobile polymer films and biophysical protocols. I would also like
to express my gratitude to Sandipan Dawn, Jacob John and Professor Ken Carter
for their encouragement and critical role toward the fabrication of substrates with
spatially arranged fibronectin dots. In addition, I would like to thank Alison Douglas
(Barker group, Georgia Tech) and Katrina Adlerz (Aranda-Espinoza group, Univer-
sity of Maryland) for additional cell-film experiments that enhanced my intuition on
the mechano-sensing of film mobility.
Furthermore, I wish to express my sincere gratitude to those who shared parts of
their life with me in Amherst and USA. I own them the feeling of being surrounded
by people who care about me. I am especially grateful to Professor Mountziaris for
his generosity and mentorship in various aspects of my life.
Finally, I want to acknowledge my extended family back home and specifically my
parents and my sister for their altruistic love. Their constant support and presence
gives me the priceless right to keep pursuing my distant Ithaca.
vi
ABSTRACT
CELL ADHESION BIOPHYSICS ON DYNAMIC
POLYMER CONSTRUCTS
FEBRUARY 2015
ANDREAS P. KOUROUKLIS
DIPLOMA, NATIONAL TECHNICAL UNIVERSITY OF ATHENS
Ph.D., UNIVERSITY OF MASSACHUSETTS AMHERST
Directed by: Professor Harry Bermudez
The biophysical characteristics of cell adhesion from single protein to cell length
scales have primarily been studied using purely elastic substrates. However, natu-
ral extracellular matrix (ECM) is viscoelastic and contains mobile components. In
this work, we combined chemistry and cell biology tools to design and characterize
laterally mobile viscoelastic polymer films that promote receptor-specific cell adhe-
sion. Moreover, we used amphiphilic block copolymers that are end-labeled with
RGD peptide ligands to allow for integrin-mediated cell adhesion. The addition of
a trace hydrophobic homopolymer in the supported bilayer block-copolymer films is
used to tune the lateral mobility of the films. NIH 3T3 fibroblasts demonstrate a non-
linear spreading response against the mobility of the RGD-displaying polymer films.
Employing immunostaining and adhesion strength assays, we decoupled the partial
contributions of focal adhesions (FA) and integrin-RGD complexes on cell adhesion.
vii
Furthermore, we employed these biomimetic polymer platforms to investigate the im-
portanceofviscousdissipationwithintheextracellularsubstrateanditsconnectionto
cell-surface receptors. Our results suggest that cells preferentially use α β and α β
v 3 5 1
integrins to control spreading and polarization in response to mechanical properties
of their substrate. In order to further control the spatial presentation of biochemical
molecules on mechanically-tunable polymer substrates, we successfully transferred fi-
bronectin patterns on bilayer polymer films. We showed that NIH 3T3 fibroblasts
spreading and adhesion features depend on the mechanical properties of these hy-
brid materials even in the presence of spatially and chemically identical biochemical
signals. Overall, the present work demonstrates the potential of amphiphilic block
copolymers to form artificial substrates that can capture a key feature of cell-ECM
interactions: specifically, the ability of cells to induce changes in the substrate over
time. Furthermore, it highlights the need for future studies on cell-substrate interac-
tions that simultaneously consider the time-dependent mechanical properties of the
ECM, the spatial characteristics of ligand presentation, and the receptor-mediated
intracellular signaling.
viii
TABLE OF CONTENTS
Page
ACKNOWLEDGMENTS ............................................. v
ABSTRACT......................................................... vii
LIST OF TABLES .................................................... xi
LIST OF FIGURES.................................................. xii
CHAPTER
1. INTRODUCTION ................................................. 1
1.1 Extracellular matrix (ECM) and cell adhesion ........................1
1.2 Biomaterials for artificial ECM mimics ..............................3
1.3 Block copolymers.................................................9
1.4 Fabrication and characterization of supported polymer films ............9
1.5 Outline ........................................................11
2. CELL ADHESION ON MOBILE POLYMER FILMS ............. 14
2.1 Introduction ....................................................14
2.2 Results.........................................................17
2.2.1 Interfacial studies .........................................17
2.2.2 Measurement of lateral mobility in supported polymer
films..................................................20
2.2.3 Cell spreading response to substrate mobility .................24
2.2.4 Focal adhesion responses to substrate mobility ................26
2.2.5 Contractility-independent mechanisms of cell adhesion .........28
2.3 Discussion ......................................................31
2.3.1 Substrate mobility as a step towards mimicry of the ECM ......31
2.3.2 Distinct modes of cell spreading due to substrate mobility ......32
2.4 Conclusions.....................................................33
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